TGF-β1对MCF-7趋化因子受体CXCR4、CCR7表达的影响
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摘要
【目的】恶性肿瘤最重要的生物学特性是侵袭和转移,这两种特性是导致肿瘤死亡的主要原因。目前研究表明,TGF-β和趋化因子及其受体参与肿瘤细胞的侵袭和转移,但在肿瘤细胞的侵袭和转移过程中,TGF-β与趋化因子及其受体间是否存在关联以及其详细机制尚无报道。本课题拟探讨TGF-β1对MCF-7细胞趋化因子受体CXCR4和CCR7表达的影响。
【方法和结果】
一、重组人TGF-β1对乳腺癌细胞系MCF-7 CXCR4、CCR7表达的影响用1,10,100ng/ml三种浓度的rhTGF-β1刺激MCF-7细胞48h后1.乳腺癌细胞系MCF-7 CXCR4的表达
1)mRNA表达:RT-PCR检测结果表明,实验组MCF-7细胞CXCR4 mRNA表达水平增高;实时定量PCR结果显示,与未刺激组相比,1ng/ml和10ng/ml的rhTGF-β1能使CXCR4的mRNA表达水平增加6倍左右,100ng/ml上调约10倍;
2)蛋白水平的表达:与空载体对照组(16.59%)相比,流式细胞仪结果显示,用不同浓度rhTGF-β1处理后MCF-7细胞表面CXCR4的表达均升高,且呈浓度依赖性的方式上调CXCR4的表达,1ng/ml,10ng/ml和100ng/ml刺激组CXCR4的表达依次为20.67%,24.60%和47.68%;
3)趋化作用:经rhTGF-β1刺激后,SDF-1α(CXCR4的配体)对刺激后的MCF-7趋化作用增强。
2.乳腺癌细胞系MCF-7 CCR7的表达
以上处理对CCR7的表达无明显影响。
二、可溶性II型TGF-β受体(Fc:TβRII)对乳腺癌细胞系MCF-7 CXCR4、CCR7表达的影响
(一)可溶性II型TGF-β受体(Fc:TβRII)真核表达载体的构建与表达
1.可溶性II型TGF-β受体(Fc:TβRII)真核表达载体的构建通过PCR方法从pH2-3FF1(含人TGF-β受体Ⅱ全长序列)扩增编码TGF-β受体Ⅱ胞外功能区的基因,将其克隆入真核表达载体pcDNA3.1-hIg 2(含人IgG Fc段)中,命名为pcDNA3.1-TβRII-hIg。经双酶切鉴定及测序分析证实,所克隆和构建的人TβRII-hIg阅读框及连接部位序列正确,表明可溶性TGF-β受体Ⅱ胞外段与人IgG Fc融合基因(TβRII-hIg)的重组真核表达质粒构建成功。
为了方便观察真核转染是否成功以及转染效率,将融合基因亚克隆至真核表达载体pIRES2-EGFP中,经双酶切鉴定及测序分析证实,成功构建了含目的基因TβRII-hIg和EGFP的双表达载体pIRES2-EGFP-TβRII-hIg。
2.可溶性II型TGF-β受体(Fc:TβRII)真核表达载体的表达采用Lipofectamine TM 2000将pIRES2-EGFP-TβRII-hIg载体转染MCF-7细胞,通过以下方法检测外源融合基因的表达
1)利用倒置荧光显微镜观察示踪蛋白EGFP,结果显示转染细胞有绿色荧光蛋白的表达,提示转染成功;
2)RT-PCR产物经琼脂糖凝胶电泳可见约1200bp的特异性条带,与目的片段一致;
3)利用抗人IgG抗体检测转染目的基因的MCF-7细胞总蛋白,Western blot结果显示分子量约41kD的特异性条带,与预期结果一致;
4)ELISA检测MCF-7细胞培养上清中存在Fc:TβRII融合蛋白,并用人IgG作为标准品对Fc:TβRII进行定量检测。结果表明转染pIRES2-EGFP-TβRII-hIg的MCF-7细胞,细胞培养上清中融合蛋白表达量到第3天约为80ng/ml。
(二)可溶性II型TGF-β受体(Fc:TβRII)对乳腺癌细胞系MCF-7 CXCR4、CCR7表达的影响
将真核表达载体pIRES2-EGFP-TβRII-hIg转染MCF-7细胞48h后1. CXCR4 mRNA的表达
RT-PCR检测结果表明,转染pIRES2-EGFP-TβRII-hIg的MCF-7细胞CXCR4mRNA的表达降低;实时定量PCR结果提示,转染pIRES2-EGFP-TβRII-hIg细胞组CXCR4 mRNA的表达显著降低,相当于pIRES2-EGFP载体组的17%左右。
2. CXCR4蛋白的表达
流式细胞仪分析结果显示,转染pIRES2-EGFP-TβRII-hIg的MCF-7细胞表面CXCR4表达显著低于pIRES2-EGFP组,分别为21.45%和31.8%。
3.趋化功能
趋化试验结果提示分泌性表达的融合蛋白Fc:TβRII可抑制SDF-1α对MCF-7细胞的趋化作用。
4.乳腺癌细胞系MCF-7 CCR7的表达
将真核表达载体pIRES2-EGFP-TβRII-hIg转染MCF-7细胞48h后,对CCR7的表达未见明显影响。
三、CXCR4启动子萤火虫荧光素酶报告基因表达载体的构建与顺式作用元件的性质分析
1.一系列5’端截短的CXCR4启动子萤火虫荧光素酶报告基因表达载体的构建提取外周血淋巴细胞基因组DNA,以其为模板,通过PCR扩增一系列5’端不同,而3’端相同的CXCR4启动子序列,将其克隆入无启动子的萤火虫荧光素酶报告基因的表达载体pGL3-basic中。经双酶切鉴定及测序分析证实,所克隆和构建的截短的人CXCR4启动子序列正确,表明构建成功。
2.缺失试验分析CXCR4启动子-231bp区域的顺式作用元件将构建的一系列5’端截短的CXCR4启动子萤火虫荧光素酶报告基因表达载体分别与作为内参的pRL-TK(海肾荧光素酶报告基因表达载体)共转染MCF-7。通过检测萤火虫荧光素酶及海肾荧光素酶的表达活性,探索CXCR4启动子-231bp区域顺式作用元件的性质。结果提示从hcxcr4启动子上游距离转录启始位点194bp(-194)到-168之间,有发挥正向调控的顺式作用元件;紧接着有一个很强的负向调控元件存在于-168bp到-160bp之间;从-160bp到-75bp的区间内又有正向调控的顺式作用元件,为进一步对CXCR4表达调控机制研究奠定了良好的基础。
【结论】以上结果表明,重组人TGF-β1刺激可上调乳腺癌细胞系MCF-7 CXCR4的表达;pIRES2-EGFP-TβRII-hIg真核表达载体转染MCF-7分泌性表达融合蛋白Fc:TβRII能下调乳腺癌细胞系MCF-7 CXCR4的表达;进一步成功构建了一系列5’端截短的CXCR4启动子萤火虫荧光素酶报告基因的表达载体,并分析了CXCR4启动子-231bp区域的顺式作用元件,为进一步对CXCR4表达调控机制研究奠定了良好的基础。
The most important biological characteristics of malignant tumour cells are the ability of invasion and migration, which are main reasons lead to death. Previous work has already revealed both transforming growth factor (TGF)-βsignaling system and chemokine-chemokine receptor are involve in tumor metastases. However, it is unclear that there is an relation between TGF-βsignaling system and chemokine-chemokine receptor during the process of tumor migration. In this research, TGF-β1-regulated CXCR4 expression was investigated in human breast cancer cell line MCF-7s.
ⅠEffcet of human recombination TGF-β1 (rhTGF-β1) on the expression of CXCR4 and CCR7 in MCF-7s
To investigate whether TGF-β1 affects CXCR4 expression in MCF-7 cells, we cultured cells with 1, 10, 100 ng/ml human recombination TGF-β1 (rhTGF-β1) for 24 hours,then detected:
1. the expression of CXCR4 in MCF-7s
1)at the mRNA level:
The experiment MCF-7s had a higher expression of CXCR4 transcripts in compared with those unstimulated cells (semiquantitative RT-PCR). The expression of CXCR4 transcripts of MCF-7s were further checked by real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR) usingβ-actin mRNA as the control for cDNA input. The quantitative RT-PCR tests confirmed that the levels of CXCR4 transcripts were higher 6-, 6-, and 10-fold in the MCF-7s treated with 1, 10, 100 ng/ml rhTGF-β1, respectively, than in those unstimulated cells.
2) at the protein level:
MCF-7s were treated with rhTGF-β1 for 48 hours, and the expression of CXCR4 was analyzed by flow cytometry(FCM). TGF-β1induced or up-regulated the surface expression of CXCR4 and in a dose-dependent manner in MCF-7s.
3)chemotaxis assay:
The functional activity of CXCR4 receptor on the surface of MCF-7s to the respective ligand (SDF-1α) was assessed by applying recombinant human SDF-1αin a cell migration assay. MCF-7s treated with rhTGF-β1 had exhibited an efficient response to SDF-1αcompared with untreated cells.
2. Effcet on the expression of CCR7 in MCF-7s treated with rhTGF-β1 It had no effect on the expression of CCR7 on MCF-7s treated with rhTGF-β1.ⅡEffcet of a soluble fusion protein(Fc:TβRII) on the expression of CXCR4 and CCR7 in MCF-7s
1. Construction of an eukaryotic expression vector pIRES2-EGFP-TβRII-hIg To construct an eukaryotic expression plasmid of human soluble TGFβRII:human IgG1 Fc (Fc: TβRII) fusion gene, a portion of the extracellular domain of the human TGF-βtype II receptor was amplified by PCR from H2-3FF. Then the receptor fragments were cloned into the expression vector pcDNA3.1-hIg. Furthermore, human tβRII:Fc sequences were subcloned into the expression vector pIRES2-EGFP and the construct was termed pIRES2-EGFP-TβRII-hIg. Finally, authenticity was confirmed by sequencing.
2. Expression of Fc:TβRII in MCF-7s
To examine the expression of Fc:TβRII, MCF-7 cells was transfected with pIRES2-EGFP-TβRII-hIg, and then the recombinant protein Fc:TβRII was expressed in the transfected cells and secreted into the medium confirmed by RT-PCR, western blotting and ELISA.
Forty-eight hours later, total cellular RNA and the cell lysate samples were collected, and subsequently mRNA transcription were analyzed by reverse transcription- PCR. As expected, we can observe the～1200bp bands in pIRES2-EGFP-TβRII-hIg transfected cells, but no band was detected in parental and transfected cells with the empty vector. This demonstrated mRNA transcription of the Fc:TβRII encoding genes in transfected cells. Furthermore, the expression of Fc:TβRII was evaluated by western blotting using anti-hIgG polyclonal antibody, and the results suggested that the Fc:TβRII protein, characterized as expected by an apparent molecular weight of 41,000 Da, was present in pIRES2-EGFP-TβRII-hIg transfected cells. However in parental cells and empty vector transfected cells no Fc:TβRII protein can be seen. Finally, a soluble typeII TGF-βreceptor secreted by experiment MCF-7s was confirmed by ELISA, and the concentration of soluble Fc:TβRII in culture supernatant was ranged from 20 ng/ml to 80 ng/ml in the first 3 days.
Together with RT-PCR , western blotting and ELISA experiments results indicated that fusion protein Fc:TβRII was secreted expression in pIRES2-EGFP-TβRII-hIg transfected MCF-7s.
3. Effcet of Fc:TβRII on the expression of CXCR4 and CCR7 in MCF-7s To examine the effect of Fc:TβRII on CXCR4 expression, MCF-7s were transfected with pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg for 48 hours,then tested
1. the expression of CXCR4 in MCF-7s
1)at the mRNA level:
cDNA samples from MCF-7s were then analyzed for CXCR4 mRNA expression by qRT-PCR. Cells secreted Fc:TβRII showed a significantly lower level of CXCR4 expression as compared with pIRES2-EGFP transfected cells. In detail, MCF-7s transfected with pIRES2-EGFP-TβRII-hIg resulted in a -2.54±0.34-fold decrease compared with control cells.
2) at the protein level:
We did FCM analysis for the change of cell surface CXCR4 expression in MCF-7s transfected with pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg. These secreted Fc:TβRII cells lost expression of CXCR4 after 48 hours transfection.
3) the expression of CCR7 in MCF-7s:
There is no significant difference in the expression of CCR7 between the two kinds of transfected cells could be detected.
4) chemotaxis assay:
We also evaluate the migration responses of MCF-7s transfected either pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg . The data showed that MCF-7 cells transfected with pIRES2-EGFP-TβRII-hIg decreased numbers of migratory cells in response to SDF-1αcompared with control cells.
ⅢConstruction of luciferase reporter and and dual luciferase assay 1.Promotor cloning and luciferase reporter construction
CXCR4 promoter sequences were amplified by PCR from human genome extracted from lympholeukocyte of human peripheral blood. A series of CXCR4 promoter fragments with different 5’ends (nt -231, -194, -179, -168, -160, -75, -63) and a common 3’end (+65), relative to the transcription start site, respectively, was ligated into the pGL3 promoterless plasmid (pGL3-basic) and validated them by DNA sequencing.
2.5' Sequences controlling human CXCR4 gene transcription.
To identify regions with transcriptional activity of CXCR4, cells were transfected with pGL3-basic vector that contains the luciferase gene after the successive 5' deletions of CXCR4 promoter and the pRL-TK vector encoding Renilla luciferase as a control for transfection efficiency. After 24 hours, luciferase assays were performed using the dual luciferase assay system.The functional expression studies indicated that the region upstream of the human cxcr4 minimal promoter contains a complex array of both positive and negative transcription control elements. Studies are in progress to identify the functional elements in this region and to determine if they could control cxcr4 gene expression by TGF-β1.
Conclusion.
Our studies revealed that MCF-7s treated with rhTGF-β1 could up-regulate the surface expression of CXCR4, while fusion protein Fc:TβRII secreted by MCF-7s transfected pIRES2-EGFP-TβRII-hIg could reduce the expression of CXCR4.
Furthermore, we constructed a series of 5' truncations of CXCR4 promoter driving the expression of a luciferase gene, then identified the regions between -231bp and transcriptional start site with transcriptional activity of CXCR4
【方法和结果】
一、重组人TGF-β1对乳腺癌细胞系MCF-7 CXCR4、CCR7表达的影响用1,10,100ng/ml三种浓度的rhTGF-β1刺激MCF-7细胞48h后1.乳腺癌细胞系MCF-7 CXCR4的表达
1)mRNA表达:RT-PCR检测结果表明,实验组MCF-7细胞CXCR4 mRNA表达水平增高;实时定量PCR结果显示,与未刺激组相比,1ng/ml和10ng/ml的rhTGF-β1能使CXCR4的mRNA表达水平增加6倍左右,100ng/ml上调约10倍;
2)蛋白水平的表达:与空载体对照组(16.59%)相比,流式细胞仪结果显示,用不同浓度rhTGF-β1处理后MCF-7细胞表面CXCR4的表达均升高,且呈浓度依赖性的方式上调CXCR4的表达,1ng/ml,10ng/ml和100ng/ml刺激组CXCR4的表达依次为20.67%,24.60%和47.68%;
3)趋化作用:经rhTGF-β1刺激后,SDF-1α(CXCR4的配体)对刺激后的MCF-7趋化作用增强。
2.乳腺癌细胞系MCF-7 CCR7的表达
以上处理对CCR7的表达无明显影响。
二、可溶性II型TGF-β受体(Fc:TβRII)对乳腺癌细胞系MCF-7 CXCR4、CCR7表达的影响
(一)可溶性II型TGF-β受体(Fc:TβRII)真核表达载体的构建与表达
1.可溶性II型TGF-β受体(Fc:TβRII)真核表达载体的构建通过PCR方法从pH2-3FF1(含人TGF-β受体Ⅱ全长序列)扩增编码TGF-β受体Ⅱ胞外功能区的基因,将其克隆入真核表达载体pcDNA3.1-hIg 2(含人IgG Fc段)中,命名为pcDNA3.1-TβRII-hIg。经双酶切鉴定及测序分析证实,所克隆和构建的人TβRII-hIg阅读框及连接部位序列正确,表明可溶性TGF-β受体Ⅱ胞外段与人IgG Fc融合基因(TβRII-hIg)的重组真核表达质粒构建成功。
为了方便观察真核转染是否成功以及转染效率,将融合基因亚克隆至真核表达载体pIRES2-EGFP中,经双酶切鉴定及测序分析证实,成功构建了含目的基因TβRII-hIg和EGFP的双表达载体pIRES2-EGFP-TβRII-hIg。
2.可溶性II型TGF-β受体(Fc:TβRII)真核表达载体的表达采用Lipofectamine TM 2000将pIRES2-EGFP-TβRII-hIg载体转染MCF-7细胞,通过以下方法检测外源融合基因的表达
1)利用倒置荧光显微镜观察示踪蛋白EGFP,结果显示转染细胞有绿色荧光蛋白的表达,提示转染成功;
2)RT-PCR产物经琼脂糖凝胶电泳可见约1200bp的特异性条带,与目的片段一致;
3)利用抗人IgG抗体检测转染目的基因的MCF-7细胞总蛋白,Western blot结果显示分子量约41kD的特异性条带,与预期结果一致;
4)ELISA检测MCF-7细胞培养上清中存在Fc:TβRII融合蛋白,并用人IgG作为标准品对Fc:TβRII进行定量检测。结果表明转染pIRES2-EGFP-TβRII-hIg的MCF-7细胞,细胞培养上清中融合蛋白表达量到第3天约为80ng/ml。
(二)可溶性II型TGF-β受体(Fc:TβRII)对乳腺癌细胞系MCF-7 CXCR4、CCR7表达的影响
将真核表达载体pIRES2-EGFP-TβRII-hIg转染MCF-7细胞48h后1. CXCR4 mRNA的表达
RT-PCR检测结果表明,转染pIRES2-EGFP-TβRII-hIg的MCF-7细胞CXCR4mRNA的表达降低;实时定量PCR结果提示,转染pIRES2-EGFP-TβRII-hIg细胞组CXCR4 mRNA的表达显著降低,相当于pIRES2-EGFP载体组的17%左右。
2. CXCR4蛋白的表达
流式细胞仪分析结果显示,转染pIRES2-EGFP-TβRII-hIg的MCF-7细胞表面CXCR4表达显著低于pIRES2-EGFP组,分别为21.45%和31.8%。
3.趋化功能
趋化试验结果提示分泌性表达的融合蛋白Fc:TβRII可抑制SDF-1α对MCF-7细胞的趋化作用。
4.乳腺癌细胞系MCF-7 CCR7的表达
将真核表达载体pIRES2-EGFP-TβRII-hIg转染MCF-7细胞48h后,对CCR7的表达未见明显影响。
三、CXCR4启动子萤火虫荧光素酶报告基因表达载体的构建与顺式作用元件的性质分析
1.一系列5’端截短的CXCR4启动子萤火虫荧光素酶报告基因表达载体的构建提取外周血淋巴细胞基因组DNA,以其为模板,通过PCR扩增一系列5’端不同,而3’端相同的CXCR4启动子序列,将其克隆入无启动子的萤火虫荧光素酶报告基因的表达载体pGL3-basic中。经双酶切鉴定及测序分析证实,所克隆和构建的截短的人CXCR4启动子序列正确,表明构建成功。
2.缺失试验分析CXCR4启动子-231bp区域的顺式作用元件将构建的一系列5’端截短的CXCR4启动子萤火虫荧光素酶报告基因表达载体分别与作为内参的pRL-TK(海肾荧光素酶报告基因表达载体)共转染MCF-7。通过检测萤火虫荧光素酶及海肾荧光素酶的表达活性,探索CXCR4启动子-231bp区域顺式作用元件的性质。结果提示从hcxcr4启动子上游距离转录启始位点194bp(-194)到-168之间,有发挥正向调控的顺式作用元件;紧接着有一个很强的负向调控元件存在于-168bp到-160bp之间;从-160bp到-75bp的区间内又有正向调控的顺式作用元件,为进一步对CXCR4表达调控机制研究奠定了良好的基础。
【结论】以上结果表明,重组人TGF-β1刺激可上调乳腺癌细胞系MCF-7 CXCR4的表达;pIRES2-EGFP-TβRII-hIg真核表达载体转染MCF-7分泌性表达融合蛋白Fc:TβRII能下调乳腺癌细胞系MCF-7 CXCR4的表达;进一步成功构建了一系列5’端截短的CXCR4启动子萤火虫荧光素酶报告基因的表达载体,并分析了CXCR4启动子-231bp区域的顺式作用元件,为进一步对CXCR4表达调控机制研究奠定了良好的基础。
The most important biological characteristics of malignant tumour cells are the ability of invasion and migration, which are main reasons lead to death. Previous work has already revealed both transforming growth factor (TGF)-βsignaling system and chemokine-chemokine receptor are involve in tumor metastases. However, it is unclear that there is an relation between TGF-βsignaling system and chemokine-chemokine receptor during the process of tumor migration. In this research, TGF-β1-regulated CXCR4 expression was investigated in human breast cancer cell line MCF-7s.
ⅠEffcet of human recombination TGF-β1 (rhTGF-β1) on the expression of CXCR4 and CCR7 in MCF-7s
To investigate whether TGF-β1 affects CXCR4 expression in MCF-7 cells, we cultured cells with 1, 10, 100 ng/ml human recombination TGF-β1 (rhTGF-β1) for 24 hours,then detected:
1. the expression of CXCR4 in MCF-7s
1)at the mRNA level:
The experiment MCF-7s had a higher expression of CXCR4 transcripts in compared with those unstimulated cells (semiquantitative RT-PCR). The expression of CXCR4 transcripts of MCF-7s were further checked by real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR) usingβ-actin mRNA as the control for cDNA input. The quantitative RT-PCR tests confirmed that the levels of CXCR4 transcripts were higher 6-, 6-, and 10-fold in the MCF-7s treated with 1, 10, 100 ng/ml rhTGF-β1, respectively, than in those unstimulated cells.
2) at the protein level:
MCF-7s were treated with rhTGF-β1 for 48 hours, and the expression of CXCR4 was analyzed by flow cytometry(FCM). TGF-β1induced or up-regulated the surface expression of CXCR4 and in a dose-dependent manner in MCF-7s.
3)chemotaxis assay:
The functional activity of CXCR4 receptor on the surface of MCF-7s to the respective ligand (SDF-1α) was assessed by applying recombinant human SDF-1αin a cell migration assay. MCF-7s treated with rhTGF-β1 had exhibited an efficient response to SDF-1αcompared with untreated cells.
2. Effcet on the expression of CCR7 in MCF-7s treated with rhTGF-β1 It had no effect on the expression of CCR7 on MCF-7s treated with rhTGF-β1.ⅡEffcet of a soluble fusion protein(Fc:TβRII) on the expression of CXCR4 and CCR7 in MCF-7s
1. Construction of an eukaryotic expression vector pIRES2-EGFP-TβRII-hIg To construct an eukaryotic expression plasmid of human soluble TGFβRII:human IgG1 Fc (Fc: TβRII) fusion gene, a portion of the extracellular domain of the human TGF-βtype II receptor was amplified by PCR from H2-3FF. Then the receptor fragments were cloned into the expression vector pcDNA3.1-hIg. Furthermore, human tβRII:Fc sequences were subcloned into the expression vector pIRES2-EGFP and the construct was termed pIRES2-EGFP-TβRII-hIg. Finally, authenticity was confirmed by sequencing.
2. Expression of Fc:TβRII in MCF-7s
To examine the expression of Fc:TβRII, MCF-7 cells was transfected with pIRES2-EGFP-TβRII-hIg, and then the recombinant protein Fc:TβRII was expressed in the transfected cells and secreted into the medium confirmed by RT-PCR, western blotting and ELISA.
Forty-eight hours later, total cellular RNA and the cell lysate samples were collected, and subsequently mRNA transcription were analyzed by reverse transcription- PCR. As expected, we can observe the～1200bp bands in pIRES2-EGFP-TβRII-hIg transfected cells, but no band was detected in parental and transfected cells with the empty vector. This demonstrated mRNA transcription of the Fc:TβRII encoding genes in transfected cells. Furthermore, the expression of Fc:TβRII was evaluated by western blotting using anti-hIgG polyclonal antibody, and the results suggested that the Fc:TβRII protein, characterized as expected by an apparent molecular weight of 41,000 Da, was present in pIRES2-EGFP-TβRII-hIg transfected cells. However in parental cells and empty vector transfected cells no Fc:TβRII protein can be seen. Finally, a soluble typeII TGF-βreceptor secreted by experiment MCF-7s was confirmed by ELISA, and the concentration of soluble Fc:TβRII in culture supernatant was ranged from 20 ng/ml to 80 ng/ml in the first 3 days.
Together with RT-PCR , western blotting and ELISA experiments results indicated that fusion protein Fc:TβRII was secreted expression in pIRES2-EGFP-TβRII-hIg transfected MCF-7s.
3. Effcet of Fc:TβRII on the expression of CXCR4 and CCR7 in MCF-7s To examine the effect of Fc:TβRII on CXCR4 expression, MCF-7s were transfected with pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg for 48 hours,then tested
1. the expression of CXCR4 in MCF-7s
1)at the mRNA level:
cDNA samples from MCF-7s were then analyzed for CXCR4 mRNA expression by qRT-PCR. Cells secreted Fc:TβRII showed a significantly lower level of CXCR4 expression as compared with pIRES2-EGFP transfected cells. In detail, MCF-7s transfected with pIRES2-EGFP-TβRII-hIg resulted in a -2.54±0.34-fold decrease compared with control cells.
2) at the protein level:
We did FCM analysis for the change of cell surface CXCR4 expression in MCF-7s transfected with pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg. These secreted Fc:TβRII cells lost expression of CXCR4 after 48 hours transfection.
3) the expression of CCR7 in MCF-7s:
There is no significant difference in the expression of CCR7 between the two kinds of transfected cells could be detected.
4) chemotaxis assay:
We also evaluate the migration responses of MCF-7s transfected either pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg . The data showed that MCF-7 cells transfected with pIRES2-EGFP-TβRII-hIg decreased numbers of migratory cells in response to SDF-1αcompared with control cells.
ⅢConstruction of luciferase reporter and and dual luciferase assay 1.Promotor cloning and luciferase reporter construction
CXCR4 promoter sequences were amplified by PCR from human genome extracted from lympholeukocyte of human peripheral blood. A series of CXCR4 promoter fragments with different 5’ends (nt -231, -194, -179, -168, -160, -75, -63) and a common 3’end (+65), relative to the transcription start site, respectively, was ligated into the pGL3 promoterless plasmid (pGL3-basic) and validated them by DNA sequencing.
2.5' Sequences controlling human CXCR4 gene transcription.
To identify regions with transcriptional activity of CXCR4, cells were transfected with pGL3-basic vector that contains the luciferase gene after the successive 5' deletions of CXCR4 promoter and the pRL-TK vector encoding Renilla luciferase as a control for transfection efficiency. After 24 hours, luciferase assays were performed using the dual luciferase assay system.The functional expression studies indicated that the region upstream of the human cxcr4 minimal promoter contains a complex array of both positive and negative transcription control elements. Studies are in progress to identify the functional elements in this region and to determine if they could control cxcr4 gene expression by TGF-β1.
Conclusion.
Our studies revealed that MCF-7s treated with rhTGF-β1 could up-regulate the surface expression of CXCR4, while fusion protein Fc:TβRII secreted by MCF-7s transfected pIRES2-EGFP-TβRII-hIg could reduce the expression of CXCR4.
Furthermore, we constructed a series of 5' truncations of CXCR4 promoter driving the expression of a luciferase gene, then identified the regions between -231bp and transcriptional start site with transcriptional activity of CXCR4
引文
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37.Muraoka, R. S., et al. Blockade of TGF-βinhibits mammary tumor cell viability, migration, and metastases. J. Clin. Invest. 2002, 109: 1551–1559.
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39.Yin, J. J., et al. TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. Journal of Clinical Investigation. 1999,103: 197-206.
40. Buckley, C.D., et al. Persistent Induction of the Chemokine Receptor CXCR4 by TGF-β1 on Synovial T Cells Contributes to Their Accumulation Within the Rheumatoid Synovium. The Journal of Immunology. 2000, 165: 3423–3429.
41. Sato, K., et al. TGF-β1 reciprocally controls chemotaxis of human peripheralblood monocyte-derived dendritic cells via chemokine receptors. The Journal of Immunology. 2000, 164: 2285–2295.
42. Ruzek, M. C. et al. Minimal effects on immune parameters following chronic anti-TGF-βmonoclonal antibody administration to normal mice. Immunopharmacol. Immunotoxicol. 2003, 25: 235–257.
43. Benigni, A. et al. Add-on anti-TGF-βantibody to ACE inhibitor arrests progressive diabetic nephropathy in the rat. J. Am. Soc. Nephrology. 2003, 14: 1816–1824.
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46. Docagne F., et al. A soluble transforming growth factor-β(TGF-β) type I receptor mimics TGF-βresponses. The Journal of Biological Chemistry 2001,276(49):46243–46250.
47. Muraoka R.S., et al. Increased malignancy of Neu-induced mammary tumors overexpressing active transforming growth factorβ1. Mol Cell Biol 2003; 23: 8691–8703.
48. Komesli S., et al. Chimeric extracellular domain of type II transforming growth factor (TGF)-βreceptor fused to the Fc region of human immunoglobulin as a TGF-βantagonist. Eur. J. Biochem. 1998, 254, 505-513
49. Rabbani Z. N., et al. Soluble TGFβtype II receptor gene therapy ameliorates acute radiation–induced pulmonary injury in rats. Int. J. Radiation Oncology Biol. Phys. 2003, 57(2): 563–572
50. Rowland-Goldsmith M. A., et al. Soluble type II transforming growth factor-β(TGF-β) receptor inhibits TGF-βsignaling in COLO-357 pancreatic cancer cells in Vitro and attenuates tumor formation1. Clinical Cancer Research 2001,7: 2931–2940
51. Dumont N., et al. Autocrine transforming growth factor-βsignaling mediates Smad-independent motility in human cancer cells. The Journal of Biological Chemistry. 2003, 278( 5) : 3275–3285
52. Koli K. M., Ramsey T. T., Ko Y., et al. Blockade of transforming growth factor-βsignaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells. The Journal of Biological Chemistry. 1997, 272(13): 8296–8302
53. Sun L. Z., et al. Expression of transforming growth factor-βtype III receptor suppresses tumorigenicity of human breast cancer MDA-MB-231 cells. The Journal of Biological Chemistry. 1997, 272(40): 25367–25372
54. Sugano Y., et al. Distortion of autocrine transforming growth factorβsignal accelerates malignant potential by enhancing cell growth as well as PAI-1 and VEGF production in human hepatocellular carcinoma cells. Oncogene. 2003, 22, 2309–2321
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2 . Hui Yin, et al.Pretreatment with soluble ST2 reduces warm hepatic ischemia/reperfusion injury. Biochemical and Biophysical Research Communications. 2006, 351: 940–946
3.Balkwill, F.. Cancer and the chemokine network. Nature 2004, 4: 540-50
4.Thelen, M. Dancing to the tune of chemokines. Nat Immunol, 2001, 2: 129-134.
5.Ebnet, K., et al. Molecular mechanisms that control leukocyte extravasation: the selectins and the chemokines. Histochem Cell Biol. 1999, 112(1): 1-23
6.Moore, M. A. The role of chemoattraction in cancer metastases. Bioessays 2001, 23: 674–676
7.Murphy, P. M. Chemokines and the molecular basis of cancer metastasis. N. Engl. J.Med. 2001, 345: 833–835
8.Kulbe, H., et al. The chemokine network in cancer - much more than directing cell movement. Int. J. Dev. Biol. 2004, 48: 489-496
9.Kleinhans, M. et al. Functional expression of the eotaxin receptor CCR3 in CD30+ cutaneous T-cell lymphoma.Blood. 2003, 101: 1487–1493.
10. Ishida, T. et al. Clinical significance of CCR4 expression in adult T-cell leukemia/lymphoma: its close association with skin involvement and unfavorable outcome. Clin. Cancer Res. 2003, 9: 3625–3634.
11. Manes, S. et al. CCR5 expression influences the progression of human breast cancer in a p53-dependent manner. J. Exp. Med. 2003, 198: 1381–1389 .
12. Kleeff, J. et al. Detection and localization of MIP-3α/ LARC/Exodus, a macrophage proinflammatory chemokine, and its CCR6 receptor in human pancreatic cancer. Int. J. Cancer. 1999. 81: 650–657.
13. Singh S., et al. Expression and functional role of CCR9 in prostate cancer cell migration and invasion. Clin. Cancer Res., 2004, 10: 8743 - 8750.
14. Dhawan, P. & Richmond, A. Role of CXCL1 in tumorigenesis of melanoma. J. Leukoc. Biol. 2002,72: 9–18.
15. Scotton, C. J., et al. Epithelial cancer cell migration: a role for chemokine receptors? Cancer Res. 2001, 61: 4961-4965.
16. Taichman, R. S., et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res. 2002, 62: 1832-1837.
17. Koshiba, T., et al. Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression. Clin Cancer Res. 2000, 6: 3530-3535.
18. Schimanski, C. C., et al. Effect of chemokine receptors CXCR4 and CCR7 on the metastatic behavior of human colorectal cancer. Clin. Cancer Res. 2005, 11: 1743 - 1750.
19. Zeelenberg, I. S., et al. The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases. Cancer Res. 2003, 63: 3833-3839.
20. Rempel, S. A., et al. Identification and localization of the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma. Clin Cancer Res. 2000, 6: 102-111.
21. Geminder, H., et al. A possible role for CXCR4 and its ligand, the CXC Chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma. J Immunol. 2001, 167: 4747-4757
22. Burger, M., et al. Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation and adhesion to stromal cells. Oncogene. 2003, 22: 8093-8101.
23. Schrader, A. J., et al. CXCR4/ CXCL12 expression and signalling in kidney cancer. Br J Cancer. 2002, 86: 1250-1256.
24. Hwang, J. H., et al. CXC chemokine receptor 4 expression and function in human anaplastic thyroid cancer cells. J Clin Endocrinol Metab. 2003, 88: 408-416.
25. Muller, A., et al.Involvement of chemokine receptors in breast cancer meastasis. Nature 2001;410:50-56.
26. Massagué, J. TGF-βsignal transduction. Annu. Rev. Biochem. 1998, 67: 753–791.
27. Miyazono, K. ,et al. Regulation of TGF-βsignaling and its roles in progression of tumors. Cancer Sci 2003; 94: 230–234
28.Derynck, R.,et al.TGF-βsignaling in tumor suppression and cancer progression. Nature genetics 2001, 29:117-129
29. Dijke, P. T. ,et al. New insights into TGF-β–Smad signalling. Trends in Biochemical Sciences. 2004, 29(5 ):265-73
30. Siegel, P. M.,et al. Cytostatic and apoptotic actions of TGF-βin homeostasis and cancer. Nature reviews 2003, 3: 807-820
31. Wakefield, L. M. ,et al. TGF-βsignaling: positive and negative effects on tumorigenesis. Current Opinion in Genetics & Development. 2002, 12:22–29
32.Bachman, K. E. ,et al. Duel nature of TGF-βsignaling: tumor suppressor vs. tumor promoter. Current Opinion in Oncology 2005, 17:49–54
33.Huang, X.M., et al. From TGF-βto Cancer Therapy. Current Drug Targets. 2003, 4: 243-250
34.Elliott, R. L. , et al. Role of transforming growth factor beta in human cancer. Journal of clinical oncology. 2005, 23(9): 2078-2093
35. Oft, M., et al. TGFβsignaling is necessary for carcinoma cell invasiveness and metastasis. Curr. Biol. 1998, 8: 1243–1252.
36. Hojo, M., et al. Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature 1999, 397: 530–534.
37.Muraoka, R. S., et al. Blockade of TGF-βinhibits mammary tumor cell viability, migration, and metastases. J. Clin. Invest. 2002, 109: 1551–1559.
38.Bandyopadhyay, A., et al. A soluble transforming growth factorβtype III receptor suppresses tumorigenicity and metastasis of human breast cancer MDA-MB-231 cells. Cancer Research. 1999, 59: 5041–5046
39.Yin, J. J., et al. TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. Journal of Clinical Investigation. 1999,103: 197-206.
40. Buckley, C.D., et al. Persistent Induction of the Chemokine Receptor CXCR4 by TGF-β1 on Synovial T Cells Contributes to Their Accumulation Within the Rheumatoid Synovium. The Journal of Immunology. 2000, 165: 3423–3429.
41. Sato, K., et al. TGF-β1 reciprocally controls chemotaxis of human peripheralblood monocyte-derived dendritic cells via chemokine receptors. The Journal of Immunology. 2000, 164: 2285–2295.
42. Ruzek, M. C. et al. Minimal effects on immune parameters following chronic anti-TGF-βmonoclonal antibody administration to normal mice. Immunopharmacol. Immunotoxicol. 2003, 25: 235–257.
43. Benigni, A. et al. Add-on anti-TGF-βantibody to ACE inhibitor arrests progressive diabetic nephropathy in the rat. J. Am. Soc. Nephrology. 2003, 14: 1816–1824.
44. Fakhrai, H. et al. Eradication of established intracranial rat gliomas by transforming growth factorβantisense gene therapy. Proc. Natl Acad. Sci. USA. 1996, 93: 2909–2914.
45. Liau, L. M., Fakhrai, H. & Black, K. L. Prolonged survival of rats with intracranial C6 gliomas by treatment with TGF-βantisense gene. Neurol. Res. 1998, 20:742–747.
46. Docagne F., et al. A soluble transforming growth factor-β(TGF-β) type I receptor mimics TGF-βresponses. The Journal of Biological Chemistry 2001,276(49):46243–46250.
47. Muraoka R.S., et al. Increased malignancy of Neu-induced mammary tumors overexpressing active transforming growth factorβ1. Mol Cell Biol 2003; 23: 8691–8703.
48. Komesli S., et al. Chimeric extracellular domain of type II transforming growth factor (TGF)-βreceptor fused to the Fc region of human immunoglobulin as a TGF-βantagonist. Eur. J. Biochem. 1998, 254, 505-513
49. Rabbani Z. N., et al. Soluble TGFβtype II receptor gene therapy ameliorates acute radiation–induced pulmonary injury in rats. Int. J. Radiation Oncology Biol. Phys. 2003, 57(2): 563–572
50. Rowland-Goldsmith M. A., et al. Soluble type II transforming growth factor-β(TGF-β) receptor inhibits TGF-βsignaling in COLO-357 pancreatic cancer cells in Vitro and attenuates tumor formation1. Clinical Cancer Research 2001,7: 2931–2940
51. Dumont N., et al. Autocrine transforming growth factor-βsignaling mediates Smad-independent motility in human cancer cells. The Journal of Biological Chemistry. 2003, 278( 5) : 3275–3285
52. Koli K. M., Ramsey T. T., Ko Y., et al. Blockade of transforming growth factor-βsignaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells. The Journal of Biological Chemistry. 1997, 272(13): 8296–8302
53. Sun L. Z., et al. Expression of transforming growth factor-βtype III receptor suppresses tumorigenicity of human breast cancer MDA-MB-231 cells. The Journal of Biological Chemistry. 1997, 272(40): 25367–25372
54. Sugano Y., et al. Distortion of autocrine transforming growth factorβsignal accelerates malignant potential by enhancing cell growth as well as PAI-1 and VEGF production in human hepatocellular carcinoma cells. Oncogene. 2003, 22, 2309–2321
1.Massagué, J. TGF-βsignal transduction. Annu. Rev. Biochem. 1998, 67: 753–791.
2.Dijke P. T. , Hill C. S. New insights into TGF-β–Smad signalling Trends in Biochemical Sciences. 2004, 29(5 )265-73
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